The clinical and bioinformatics analysis for the role of antihypertension drugs on mortality among patients with hypertension hospitalized with COVID‐19

Abstract Comorbidities such as hypertension could exacerbate symptoms of coronaviral disease 2019 (COVID)‐19 infection. Patients with hypertension may receive both anti‐COVID‐19 and antihypertension therapies when infected with COVID‐19. However, it is not clear how different classes of anti‐hypertension drugs impact the outcome of COVID‐19 treatment. Herein, we explore the association between the inpatient use of different classes of anti‐hypertension drugs and mortality among patients with hypertension hospitalized with COVID‐19. We totally collected data from 278 patients with hypertension diagnosed with COVID‐19 admitted to hospitals in Wuhan from February 1 to April 1, 2020. A retrospective study was conducted and single‐cell RNA‐sequencing (RNA‐Seq) analysis of treatment‐related genes was performed. The results showed that Angiotensin II receptor blocker (ARB) and calcium channel blocker (CCB) drugs significantly increased the survival rate but the use of angiotensin‐converting enzyme inhibitor/β‐block/diuretic drugs did not affect the mortality caused by COVID‐19. Based on the analysis of four public data sets of single‐cell RNA‐Seq on COVID‐19 patients, we concluded that JUN, LST1 genes may play a role in the effect of ARB on COVID‐19‐related mortality, whereas CALM1 gene may contribute to the effect of CCB on COVID‐19‐related mortality. Our results provide guidance on the selection of antihypertension drugs for hypertensive patients infected with COVID‐19.

and calcium channel blocker (CCB) drugs significantly increased the survival rate but the use of angiotensin-converting enzyme inhibitor/β-block/diuretic drugs did not affect the mortality caused by COVID-19. Based on the analysis of four public data sets of single-cell RNA-Seq on COVID-19 patients, we concluded that JUN, LST1 genes may play a role in the effect of ARB on COVID-19-related mortality, whereas CALM1 gene may contribute to the effect of CCB on COVID-19-related mortality.
Our results provide guidance on the selection of antihypertension drugs for hypertensive patients infected with COVID-19. and conventional disease medicament remain to be analyzed. 4 Hypertension is one of the most fatal diseases and threatens more than 100 million people in the last three decades worldwide. 5 It has become one of the common comorbidity with COVID-19. It has been reported that COVID-19 patients with hypertension, who suffer from a higher risk of death, have to consequently administer drugs for hypertension and COVID-19 simultaneously. However, one recent report demonstrates that angiotensin-converting enzyme inhibitors (ACEI) and Angiotensin II receptor blocker (ARB) may reduce the mortality risk of COVID-19 patients, raising the question if and how hypertension drugs interfere with virus replication in COVID-19 patients. Additionally, another concern is how the expression of genes associated with hypertension drugs is affected by SARS-CoV-2 infection, particularly in patients bearing both hypertension and  To protect these patients' health, it is worthwhile to study the effects of existing hypertension medicine upon COVID-19 pathology.
Here we analyzed the age-and sex-dependent effects of diverse hypertension drugs on COVID-19 symptoms and found that calcium channel blockers (CCBs) played a protective role in the progression of COVID-19 pathogenesis in a statistically significant manner, particularly in the patients over 70 years old. Furthermore, we first investigated the expression patterns of the hypertension drugrelated genes in COVID-19 patients by data set mining and modeling, suggesting the molecules being potentially involved in both hypertension and COVID-19 pathology, such as JUN, LST1, and SLC18A2.
Our study therefore adds essential medicinal information to COVID-19 protection. We selected 278 hypertension patients from all these cases with COVID-19 and hypertension was diagnosed as systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg three times on different days without taking antihypertensive drugs. 7 Patient demographics (age and gender), time of admission, time of discharge, therapeutic interventions of hypertension, and clinical outcomes during the hospitalization were collected from the e-health system. Hypertension treatment drugs for patients included ACEI, ARB, CCB, β-blockers, and diuretic. Recruited patients who received combination medication were considered to receive each drug in our study, separately. The following inclusion and exclusion criteria were used to identify patients in this study. The inclusion criteria were hypertensive patients diagnosed as COVID-19 aged above 35 years old, who were admitted to the hospitals mentioned above from February 1 to April 1, 2020. Patients without completed medical records were excluded (cured during the follow-up period but the cured time was not recorded/hospitalized during the follow-up period, but it was not known whether an outcome event occurred).
Informed consent was obtained from included patients or their representatives, and the study was approved by the participating hospitals' review boards.

| STATISTICAL ANALYSIS
A descriptive analysis was used to describe the characteristics of included patients. The primary endpoint was 75-day all-cause death. All eligible patients were included in the analysis of 75-day all-cause death. The Kaplan-Meier method was performed to display the survival probability and the differences in survival were analyzed by the log-rank test. A univariate Cox proportionalhazards model was conducted to have a primary analysis of the relationship between each independent variable and outcome, and statistically significant factors were taken into the multivariable analysis. Multivariable analyses with the Cox proportional hazards model were used to estimate the adjusted hazard ratio (HR) between in-hospital use of different drug therapy and all-cause mortality in patients with hypertension and hospitalized due to COVID-19. Furthermore, the multivariate Cox proportional hazards model was used for subgroup analysis for different sex groups and age groups, respectively, to explore the differences in the effects of various drug therapy in the subgroups. 70 was the age cutoff. All data analyses were conducted by R studio 3.6.2. The "survival" and "survminer" packages were used for the survival analysis and Cox regression analysis. All statistical tests were two-sided with a statistically significant p-value < 0.05.

| Single-cell RNA-sequencing (RNA-Seq) analysis
We downloaded four data sets from three publications and the downloaded data have been aligned and annotated; the R package Seurat V3 was used for filtering, standardization, conversion, dimensionality reduction, clustering, gene differential expression analysis, and visualization. The standards of filtering, clustering, and dimensionality reduction were consistent with the respective original texts. "MAST" was used for gene differential expression analysis.
The bronchoalveolar lavage fluid (BALF) single-cell RNA-Seq raw data from Liao et al. 8 including three moderate, six severe cases, and three healthy controls were downloaded from the Gene Expression Omnibus (GEO) database (accession number GSE145926). In addition, there was also a case of health control data from GEO GSM3660650. According to the original text, 66 452 cells were obtained by using the following parameters: nFeature > 200 and <6000, nCount > 1000, mitochondrial gene percentage < 10. The function "Lognormalize" was used for normalization. Then, we identified the top 2000 highly variable genes by "VST" method in the function FindVariableFeatures. The filtered cells were integrated to removing the batch effect (dim=1: 50). In ScaleData, "nCount_ RNA" and "percent. mito" has been regressed. According to the cell source, the cluster was divided into three clusters: healthy, mild, and severe. Finally, the significant genes with adjusted p < 0.05 in different groups were obtained for downstream analysis by performing gene differential expression analysis with "MAST" in FindAllMarkers function.
The peripheral blood mononuclear cells single-cell RNA-Seq data from Wilk et al. 9 including seven severe cases and six healthy controls were downloaded from the GEO database (accession number GSE150728). According to the following parameters, we filtered, normalized, and integrated the data of 14 experimental subjects (1 patient was sampled once before and after intubation): 1000 < nCount < 15 000, mitochondrial genes < 20, at least 10 genes must be expressed in each cell. Then the rest of the analyses followed the analysis of GSE145926 data set.

| RESULTS
A total of 278 patients were selected in the study ( different sources and examined the expression patterns of hypertension drug-associated genes. The gene expression profiles were extracted from the BALF, 8 peripheral blood, 9 and NS/pharyngeal swabs. 12 We found that although the transcriptome data sets were independent, the differential expressed genes related with ARB and CCB show similar patterns between the severe versus control comparisons in the COVID-19 patients ( Figure 2). JUN, LST1, and CAML1 expression were increased in COVID-19 patients.

| DISCUSSION
The highly contagious SARS-CoV-2 virus endangers human health in many aspects and has been found to impose its effects on the pathogenesis of other diseases, such as cancer, diabetes, and hypertension. 6  two ARB associated genes, JUN and LST1, have been found substantially increased in COVID-19 patients relative to controls, providing a supportive interpretation for the previous report. 6 Although the etiology of hypertension is unknown, there is evidence that the nature of hypertension is chronic inflammation.
Changes in inflammatory factors, immune cell ratios, and phenotypes can be observed in hypertensive patients. 16 JUN and LST1 are associated with inflammation and immunity. 17 JUN is a target gene of the antihypertensive drug Irbesartan and LST1 affects leukocyte abundance and T-cell proliferation. 17,18 CALM1 is a target gene of the antihypertensive drug Felodipine, which regulates calcium channels. 19 Therefore, the use of ARB or CCB treatment may decrease the expression level of related genes in infected patients, thereby improving the survival rate of COVID-19 hypertensive patients. [16][17][18][19] The question if the expression of these genes is directly regulated by SARS-CoV-2 infection still needs to be further explored. There were a few limitations in our study. Firstly, the number of included patients treated with diuretic/β-blockers was small. Second, we did not consider the ACKNOWLEDGMENT N-Y SHAO wants to thank the UM internal grant SRG2019-00177-FHS.